Abstract Excitatory amino acid transporters (EAATs) remove synaptically released glutamate and maintain extracellular glutamate concentrations below neurotoxic levels. Particularly the glial glutamate transporter EAAT2 plays a major role in glutamate clearance in synaptic clefts. Removal of excess cellular glutamate is strongly implicated as a clinically relevant means to treat neurodegenerative diseases where excitotoxicity due to excess glutamate contributes to neuronal injury and death. We have designed a unique research program to understand the molecular mechanisms of allosteric modulation of glutamate transporters by recently discovered small-compound molecules, including long-sought activators of transport. To reach this goal, we will obtain and integrate multidisciplinary knowledge of the transport function, 3D structures, and single-molecule dynamics of the transporters and their complexes with the allosteric compounds. In turn, this knowledge will aid understanding the basic membrane transport mechanisms of the most important excitatory neurotransmitter in the human brain. We will pursue the following aims: Aim 1: Elucidate the molecular determinants within the human EAATs that are important for allosteric modulator activity. We will use functional studies and computational approaches to define the allosteric site within EAAT2 that mediate the effects of the compounds. Aim 2: Determine the structures of human EAATs in complex with allosteric modulators. We will determine the three-dimensional structures of EAATs in complex with positive and negative allosteric modulators to unravel the atomic details of their coordination. Aim 3: Establish whether allosteric modulators modulate the function of EAATs though altering the rates of conformational transitions underlying transport. We will examine the conformational dynamics and its modulation by allosteric modulators using single-molecule FRET and other spectroscopic techniques and couple these studies with single-vesicle/single-transporter assays to determine the effects of allosteric modulators on turnover rates and the timing of transport cycles. IMPACT: Information generated in this research program will open new avenues for drug discovery as these transporters serve as important drug targets for many severe debilitating CNS conditions, such as traumatic brain injury, stroke, epilepsy, ALS and neuropathic pain, that collectively affect nearly 5% of the American population.